Not applicable.
The present invention is generally directed to the detection of genetic markers in a genomic system. The present invention is more specifically directed to the simultaneous amplification of multiple distinct polymorphic genetic loci using the polymerase chain reaction or other amplification systems to determine, in one reaction, the alleles of each locus contained within the multiplex system.
DNA typing is commonly used to identify the parentage of human children, and to confirm the lineage of horses, dogs, other animals, and agricultural crops. DNA typing is also commonly employed to identify the source of blood, saliva, semen, and other tissue found at a crime scenes or other sites requiring identification of human remains. DNA typing is also employed in clinical settings to determine success or failure of bone marrow transplantation and presence of particular cancerous tissues. DNA typing involves the analysis of alleles of genomic DNA with characteristics of interest, commonly referred to as xe2x80x9cmarkersxe2x80x9d. Most typing methods in use today are specifically designed to detect and analyze differences in the length and/or sequence of one or more regions of DNA markers known to appear in at least two different forms in a population. Such length and/or sequence variation is referred to as xe2x80x9cpolymorphism.xe2x80x9d Any region (i.e. xe2x80x9clocusxe2x80x9d) of DNA in which such a variation occurs is referred to as a xe2x80x9cpolymorphic locus.xe2x80x9d The methods and materials of the present invention are designed for use in the detection of multiple loci of DNA, some or all of which are polymorphic loci.
Genetic markers which are sufficiently polymorphic with respect to length or sequence have long been sought for use in identity applications, such as paternity testing and identification of tissue samples collected for forensic analysis. The discovery and development of such markers and methods for analyzing such markers have gone through several phases of development over the last several years.
The first identified DNA variant markers were simple base substitutions, i.e. simple sequence polymorphisms, which were most often detected by Southern hybridization assays. For examples of references describing the identification of such markers, designed to be used to analyze restriction endonuclease-digested DNA with radioactive probes, see: Southern, E. M. (1975), J. Mol. Biol. 98(3):503-507; Schumm, et al. (1988), American Journal of Human Genetics 42:143-159; and Wyman, A. and White, R. (1980) Proc. Natl. Acad. Sci, U.S.A. 77:6754-6758.
The next generation of markers were size variants, i.e. length polymorphisms, specifically xe2x80x9cvariable number of tandem repeatxe2x80x9d (VNTR) markers (Nakamura Y., et al. (1987), Science 235: 1616-1622; and U.S. Pat. No. 4,963,663 issued to White et al. (1990); U.S. Pat. No. 5,411,859 continuation of 4,963,663 issued to White et al. (1995)) and xe2x80x9cminisatellitexe2x80x9d markers (Jeffreys et al. (1985a), Nature 314:67-73; Jeffreys et al. (1985b) Nature 316:76-79., U.S. Pat. No. 5,175,082 for an invention by Jeffreys). Both VNTR and minisatellite markers, contain regions of nearly identical sequences repeated in tandem fashion. The core repeat sequence is 10 to 70 bases in length, with shorter core repeat sequences referred to as xe2x80x9cminisatellitexe2x80x9d repeats and longer repeats referred to as VNTRs. Different individuals in a human population contain different numbers of the repeats. The VNTR markers are generally more highly polymorphic than base substitution polymorphisms, sometimes displaying up to forty or more alleles at a single genetic locus. However, the tedious process of restriction enzyme digestion and subsequent Southern hybridization analysis are still required to detect and analyze most such markers.
The next advance involved the joining of the polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202 by Mullis, K. B.) technology with the analysis of VNTR loci (Kasai, K. et al. (1990) Journal Forensic Science 35(5):1196-1200). Amplifiable VNTR loci were discovered, which could be detected without the need for Southern transfer. The amplified products are separated through agarose or polyacrylamide gels and detected by incorporation of radioactivity during the amplification or by post-staining with silver or ethidium bromide. However, PCR can only be used to amplify relatively small DNA segments reliably, i.e. only reliably amplifying DNA segments under 3,000 bases in length Ponce, M and Micol, L. (1992) NAR 20(3):623; Decorte R, et al. (1990) DNA Cell Biol. 9(6):461-469). Consequently, very few amplifiable VNTRs have been developed.
In recent years, the discovery and development of polymorphic short tandem repeats (STRs) as genetic markers has stimulated progress in the development of linkage maps, the identification and characterization of diseased genes, and the simplification and precision of DNA typing. Specifically, with the discovery and development of polymorphic markers containing dinucleotide repeats (Litt and Luty (1989) Am J. Hum Genet 3(4):599-605; Tautz, D (1989) NAR 17:6463-6471; Weber and May (1989) Am J Hum Genet 44:388-396; German Pat. No. DE 38 34 636 C2, inventor Tautz, D; U.S. Pat. No. 5,582,979 filed by Weber, L.), STRs with repeat units of three to four nucleotides (Edwards, A., et al. (1991) Am. J. Hum. Genet. 49: 746-756.; Hammond, H. A., et al. (1994) Am. J. Hum. Genet. 55: 175-189; Fregeau, C. J.; and Fourney, R. M. (1993) BioTechniques 15(1): 100-119.; Schumm, J. W. et al. (1994) in The Fourth International Symposium on Human Identification 1993, pp. 177-187 (pub. by Promega Corp., 1994); and U.S. Pat. No. 5,364,759 by Caskey et al.; German Pat. No. DE 38 34 636 C2 by Tautz, D.) and STRs with repeat units of five to seven bases (See, e.g. Edwards et al. (1991) Nucleic Acids Res. 19:4791; Chen et al. (1993) Genomics 15(3): 621-5; Harada et al. (1994) Am. J. Hum. Genet. 55: 175-189; Comings et al. (1995), Genomics 29(2):390-6; and Utah Marker Development Group (1995), Am. J. Genet. 57:619-628; and Jurka and Pethiyagoda (1995) J. Mol. Evol. 40:120-126)), many of the deficiencies of previous methods have been overcome. STR markers are generally shorter than VNTR markers, making them better substrates for amplification than most VNTR markers.
STR loci are similar to amplifiable VNTR loci in that the amplified alleles at each such locus may be differentiated based on length variation. Generally speaking STR loci are less polymorphic at each individual locus than VNTR loci. Thus, it is desirable to amplify and detect multiple STR systems in a single amplification reaction and separation to provide information for several loci simultaneously. Systems containing several loci are called multiplex systems and many such systems containing up to 11 separate STR loci have been described. See, e.g., Proceedings: American Academy of Forensic Sciences (Feb. 9-14, 1998), Schumm, James W. et al., p. 53, B88; Id., Gibson, Sandra D. et al., p. 53, B89; Id., Lazaruk, Katherine et al., p. 51, B83; Sparkes, R. et al., Int J Legal Med (1996) 109:186-194; AmpFlSTR Profiler(trademark) PCR Amplification Kit User""s Manual (1997), pub by Perkin-Elmer Corp, i-viii and 1-1 to 1-10; AmpFlSTR Profiler Plus(trademark) PCR Amplification Kit User""s Manual (1997), pub by Perkin-Elmer Corp., i viii and 1-1 to 1-10; AmpFlSTR COfiler(trademark) PCR Amplification Kit User Bulletin (1998), pub by Perkin-Elmer Corp. i-iii and 1-1 to 1-10; 9th International Symposium on Human Identification (Oct. 7-10, 1998), pub. by Promega Corp., Staub, Rick W. et al., Poster Abstract 15; Id., Willard, Jeanne M. et al., Poster Abstract 73; and Id., Walsh, P. Sean, et al., Speaker Abstract for 8:50am-9:20am, Thursday, Oct. 8, 1998.
Amplification protocols with STR loci can be designed to produce small products, generally from 60 to 500 base pairs (bp) in length, and alleles from each locus are often contained within a range of less than 100 bp. This allows simultaneous electrophoretic analysis of several systems on the same gel or capillary electrophoresis by careful design of PCR primers such that all potential amplification products from an individual system do not overlap the range of alleles of other systems. Design of these systems is limited, in part, by the difficulty in separating multiple loci in a single gel or capillary. This occurs because there is spacial compression of fragments of different sizes, especially longer fragments in gels or capillaries, i.e., commonly used means for separation of DNA fragments by those skilled in the art.
The United States Federal Bureau of Investigation (xe2x80x9cFBIxe2x80x9d) has established and maintains a Combined DNA Index System (xe2x80x9cCODISxe2x80x9d), a database of DNA typing information. Local, state, and national law enforcement agencies use the CODIS system to match forensic DNA evidence collected at crime scenes with DNA information in the database. CODIS and other national database systems have proven to be an effective tool for such agencies to use in solving violent crimes. (See, e.g. Niezgoda, Stephen, in Cambridge Healthtech Institute""s Second Annual Conference on DNA Forensics: Science, Evidence, and Future Prospects (Nov. 17-18, 1998), pp. 1-21.; Niezgoda, Stephen in Proceedings From The Eighth International Symposium on Human Identification 1997, pub. by Promega Corporation (1998), pp 48-49; Frazier, Rachel R. E. et al. Id., pp. 56-60; Niezgoda, S. J. Profiles in DNA 1(3): 12-13; Werrett, D. J. and Sparkes, R. in Speaker Abstracts: 9th International Symposium on Human Identification (Oct. 7-10, 1998) pp. 5-6). Until recently, only restriction fragment length polymorphism (xe2x80x9cRFLPxe2x80x9d) data obtained from the analysis of particular VNTR loci was considered a core component in the database. The FBI has recently identified thirteen polymorphic STR loci for inclusion in the CODIS database. The thirteen CODIS STR loci are HUMCSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31. (Budowle, Bruce and Moretti, Tamyra in Speaker Abstracts: 9th International Symposium on Human Identification (Oct. 7-10, 1998) pp. 7-8). Both VNTR and STR marker data are currently maintained in the CODIS database. (See, e.g. Niezgoda, Stephen in Second Annual Conference on DNA Forensics, supra). Until the present invention, the number of loci which could be co-amplified in a single reaction, and analyzed thereafter was limited. Specifically, no materials or methods had been developed for use in multiplex amplification of thirteen or more STR loci, much less the thirteen polymorphic STR loci identified for use in the CODIS database.
The materials and methods of the present method are designed for use in multiplex analysis of particular polymorphic loci of DNA of various types, including single-stranded and double-stranded DNA from a variety of different sources. The present invention represents a significant improvement over existing technology, bringing increased power of discrimination, precision, and throughput to DNA profiling for linkage analysis, criminal justice, paternity testing, and other forensic, medical, and genetic identification applications.
It is, therefore, an object of the present invention to provide a method and materials for the simultaneous amplification of sets of loci, which include multiple distinct polymorphic short tandem repeat (STR) loci, in a single multiplex reaction, using PCR or other amplification systems in combination with gel electrophoresis, capillary electrophoresis or other separation and detection methods to analyze and compare the relative lengths of the alleles of each locus amplified in the multiplex reaction. Multiplex analysis of the sets of loci disclosed herein has not been previously described in the prior art. There has also not been any previous description of the sequences for many of the primers disclosed herein below, all of which are shown to be useful in multiplex amplification of the sets of loci disclosed.
It is also an object of the present invention to provide a method, a kit, and primers specific for multiplex amplifications comprising specified loci.
These and other objects are addressed by the present invention which is directed to a method and materials for simultaneously analyzing or determining the alleles present at each individual locus of each multiplex. In general, the method of this invention comprises the steps of (a) obtaining at least one DNA sample to be analyzed, wherein the DNA sample has at least thirteen loci which can be co-amplified; (b) co-amplifying the at least thirteen loci of the DNA sample; and (c) detecting the amplified materials in a fashion which reveals the polymorphic nature of the systems employed.
In one embodiment, the present invention is a method of simultaneously determining the alleles present in a set of loci from one or more DNA samples, comprising the steps of:
(a) obtaining at least one DNA sample to be analyzed;
(b) selecting a set of loci of the DNA sample, comprising at least thirteen short tandem repeat loci which can be co-amplified;
(c) co-amplifying the loci in the set in a multiplex amplification reaction, wherein the product of the reaction is a mixture of amplified alleles from each of the co-amplified loci in the set; and
(d) evaluating the amplified alleles in the mixture to determine the alleles present at each of the loci analyzed in the set within the DNA sample.
At least four of the at least thirteen short tandem repeat loci are preferably selected from the group of loci consisting of:
D3S1539, D4S2368, D5S818, D7S820, D9S930, D10S1239, D13S317, D14S118, D14S548, D14S562, D16S490, D16S539, D16S753, D17S1298, D17S1299, D19S253, D20S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13A01, HUMBFXIII, HUMLIPOL, HUMvWFA31.
In another embodiment of the invention, the set of loci selected in step (b) of In another embodiment of the invention, the set of loci selected in step (b) of the method comprises thirteen CODIS STR loci (i.e., D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31) which can be co-amplified and analyzed by themselves, or with additional loci using methods of the present invention.
In a further aspect, this invention is a kit for simultaneously analyzing a set of loci of genomic DNA, comprising oligonucleotide primers for co-amplifying a set of loci of the genomic DNA to be analyzed, wherein the set of loci comprises at least thirteen short tandem repeat loci which can be co-amplified in the same multiplex reaction, and wherein the primers are in one or more containers. More preferably, the kit comprises oligonucleotide primer pairs for co-amplifying a set of at least thirteen loci of human genomic DNA, the set of loci comprising D3S1358, D5S818, D7S820, D8S1179, D 13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31.
In yet a further aspect, the invention is primer sequences and primer pairs for amplifying specific loci of human DNA. Use of the primers and primer pairs of this invention for multiplex analysis of human DNA is demonstrated herein, below. The primers of this invention are suitable for use in the method of this invention, wherein they can be used in labeled form, as noted below, to assist the evaluation step of the method.
The approaches specified in the present invention produce savings of time, labor, and materials in the analysis of loci contained within the multiplexes. The method of the present invention allows thirteen or more, even as many as sixteen or more, loci to be co-amplified in one tube using a single amplification reaction, instead of amplifying each locus independently in separate tubes or in smaller groups of loci.
The present invention has specific use in the field of forensic analysis, paternity determination, monitoring of bone marrow transplantation, linkage mapping, and detection of genetic diseases and cancers. By allowing thirteen methods of the present invention significantly increase the certainty with which one can match DNA prepared from different samples from the same individual. The need to match or distinguish accurately between samples containing very small amounts of DNA is particularly acute in forensics applications, where many convictions (and acquittals) turn on DNA typing analysis.
Scientists, particularly forensic scientists, have long appreciated the need to analyze multiple polymorphic loci of DNA in order to ensure that a match between two samples of DNA is statistically significant. (Presley, L. A. et al., in The Third International Symposium on Human Identification 1992, pp. 245-269 (pub. by Promega Corp., 1993); Bever, R. A., et al., in The Second International Symposium on Human Identification 1991, pp.103-128. (pub. by Promega Corp., 1992)). However, until this invention, one could not simultaneously analyze thirteen or more STR loci in a single reaction. To realize the importance of such multiplexing capabilities, it helps to understand some of the mathematics behind DNA typing analysis.
For purposes of illustration, suppose every STR locus has a genotype (i.e., pattern of two alleles) frequency of one in ten. In other words, suppose that the chance of two randomly selected individuals have a matching type for a single STR is 1/10. However, if two different STR loci are analyzed, the chance of a random match with both systems is 1/100. If three STR loci are analyzed, the chances of a random match with each of the three systems is 1/1,000 and so on. Consequently, it is easy to see how increasing the number of STR loci analyzed reduces the likelihood of random matches within the general population, thereby increasing the chance one can accurately identify a suspect""s presence at a crime scene by comparing the individual""s type with crime scene evidence. Similar reasoning can be used to conclude that the method of this invention also would increase the likelihood of accurately identifying a suspected father in a paternity case, of correctly matching bone marrow tissue, of developing significant results from linkage mapping studies, and of detecting genetic diseases and cancers.
Further objects, features, and advantages of the invention will be apparent from the following best mode for carrying out the invention and the illustrative drawings.